PCR-restriction fragment length polymorphism analysis (PRA) of Mycobacterium leprae from human lepromas and from a natural case of an armadillo of Corrientes, Argentina.

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that allows identification of the species without the need for specialized equipment. Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in internal organs such as lymph nodes, spleen and liver, as well as in nerves and skin. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans. Mycobacterium leprae were recognized in those naturally infected animals through different techniques. Three samples of extracted DNA of the mycobacteria present in the spleen, liver and popliteal lymph node of a naturally infected animal during the Experimental Program in Armadillo (PEA) and three samples of human lepromas were processed by PRA. The patterns of the six samples analyzed were identical and were characteristic of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of armadillos naturally infected with the Hansen bacillus.

Toxigenic Escherichia coli isolated from pigs in Argentina.

The presence of porcine toxigenic E. coli (ETEC, VTEC) in 28 piggeries (5% of total) of the central and northeast region of Argentina was studied for a better understanding of the epidemiology of porcine strains. Samples were taken by rectal swabs from healthy piglets and from those with diarrhoea, in addition to their dams. Between 5-10 colonies were isolated from each one of 223 animals sampled from 1992 to 1997. By using specific primers each strain was screened by PCR for VT1, VT2all, VT2e, STIa, and LTI toxin genes. Only strains positive for any of the toxins mentioned above were screened for STb. Their O serogroups were determined by agglutination. All of the above enterotoxins and verocytotoxins were found in E. coli isolated from the animals. The STIa gene was detected in E. coli isolated from 27/127 piglets with diarrhoea, in comparison with LTI (4/127 pigs). No toxin gene was amplified from E. coli isolated from either healthy piglets or their dams. When strains isolated from 48 piglets without diarrhoea but showing delayed growth were analysed by PCR, their toxin profile was determined to be VT1 (1/48 piglets), VT2all (5/48), STIa (1/48), LTI (3/48) and VT2e (3/48). Serogroup O64 prevailed among ETEC; O138 prevailed for ETEC/VTEC strains. This is the first extensive study regarding porcine toxigenic E. coli in Argentina and constitutes an important database for the implementation of prevention measures.

Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America.

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.

[Colibacillosis in swine: proof of vaccine efficacy]. / Colibacilosis en lechones: prueba de eficacia de una vacuna.

The damage caused in the economy and animal sanity by the porcine colibacilosis are significant and they deserve the investigation of preventive measures that give answers to the producers. Existing at the present time approximately 21,000 pigs in Corrientes and 110,000 in Chaco provinces of Argentine, the losses for diarrhea that exterminate whole litters, acquire relevance, specially if they can be prevented or cured. For that reason, having 21 strains of enteropathogenic and verocitotoxigenic E. coli (ETEC/VTEC) isolated from pigs of the North East of Argentine, that were recognized by PCR, two were selected, containing the genes for STIa, STIb, LTI, VT2e (SLT-IIv) and F4 (K88). They were spread on nutrient agar and Minca medium, to obtain the suspension in PBS, which was inactivated with formol. After the sterility and innocuity controls, it was diluted to a 12 x 10(8) concentration to make the mouse protection test in 20 mice, of 18-20 g, inoculating the vaccine the days 1, 4, 7 and 10, by the intraperitoneal route, doses of 0.25 ml each one. The day 21 after beginning the test the animals were challenged with 50 LD50, and a protection of 85% was obtained. To determine the LD50, we prepared a suspension in physiologic solution, corresponding to Mac Farland's tube No 10, making dilutions from 10(-1) to 10(-5) and applying the statistical method of Reed and Muench. These first results encourage us to continue working after a prophylactic measure that were effective, potent and elaborated with strains of this area.

[Trichophyton simii in a "Caí" monkey (Cebus apella) colony in the province of Corrientes, Argentina.]. / Trichophyton simii en una colonia de monos "Caí" (Cebus apella), en la provincia de Corrientes, Argentina.

The objective is to describe an outbreak of Trichophyton simii in a Cebus apella monkey colony in Argentine. During summer, alopecic zones appeared on dorsal regions from head to base of the tail of the animals. The hair and skin of nine animals were streaked onto Sabouraud dextrose with cloramphenicol and incubated at 25 degrees C. By the 10th day, white, filamentous colonies, which turned pale pink, developed from simples of four animals. Microscopical examinations were carried out and, because of colony and macroconidia morphology, were classified as Trichopyton simii. Although infection with T. simii is considered a zoonosis, we did not find human cases.

Comparison of different genetic markers for molecular epidemiology of bovine tuberculosis.

In the present study three different genetic markers were used in an RFLP study to differentiate Mycobacterium bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence IS6110, the direct repeat (DR) sequence flanking IS6110, and a polymorphic GC-rich repetitive sequence (PGRS), called pMBA2. Two restriction enzymes were used, PvuII for IS6110 and DR (DR/PvuII) and AluI for DR (DR/AluI) and pMBA2. DNA from 85 of M. bovis isolates was digested with PvuII and hybridized with IS6110 and Dr. IS6110 was not useful to differentiate M. bovis because most of the isolates contain a single monomorphic copy. The use of DR allowed a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and hybridized with DR and pMBA2. In this condition these probes differentiated the isolates in many different RFLP types. By combining the patterns generated with DR/AluI and pMBA2 it was possible to increase the differentiation up to obtain 30 different RFLP types and 54% of the isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and pMBA2 could be, at the moment, the probes of choice for comparisons of M. bovis isolates in different regions and for epidemiological surveillance of bovine tuberculosis.